Phase contrast microscopy

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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georgetmacro
Posts: 37
Joined: Mon Sep 18, 2017 10:12 pm

Phase contrast microscopy

#1 Post by georgetmacro » Wed Apr 03, 2019 2:56 am

Hi. I have just purchased an inexpensive Lomo 'negative phase contrast' condenser to experiment with. The NA is 0.8 according to the supplier. The unit comes with two objectives that are supposedly specific to this type of microscopy. The item has not arrived yet so I would like to learn something about the technique beforehand. I sort of know what these instruments do but would like to understand more. e.g., why does the higher NA attract a higher price? Is it because the optics are better, bigger? Why do the instruments require a specific type of objective? I would also like to see any images people have achieved experimenting with the technique. Cheers George.

PeteM
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Joined: Tue Dec 06, 2016 6:22 am
Location: Santa Cruz, CA

Re: Phase contrast microscopy

#2 Post by PeteM » Wed Apr 03, 2019 3:16 am

Phase contrast setups require matching components. In some cases you can find condenser annuli (which project a bright ring of light through the condenser) and objective phase contrast rings (typically sort of grayed out rings inside the objective, with a different refractive index) that match. But you can't count on compatibility without testing.

This is most likely and older Lomo setup -- and its good you'll have a couple objectives that match the phase condenser. Next, you'll need a microscope capable of:

1) Holding the condenser
2) Focusing closely enough to use the short barrel Lomo objectives

I once adapted a Lomo setup to a Wesco microscope, but it was a pain. Had to raise the stage and slightly bore out the condenser holder. If you got a useful range of phase objectives with the phase condenser (for protists?), you might want to start looking for an appropriate stand

My one caution -- and you might want to post a like or a picture -- is that .80 NA is low for an upright Lomo phase condenser. Is it for an inverted scope?? Or was this the NA for one of the objectives?

georgetmacro
Posts: 37
Joined: Mon Sep 18, 2017 10:12 pm

Re: Phase contrast microscopy

#3 Post by georgetmacro » Wed Apr 03, 2019 6:55 am

PeteM.
The phase contrast instrument is, as far as I know, a standard under-stage one to suit Lomo Biolam microscopes. The mounting diameter is ~37 mm. I say approximately because the one I have on my modified Biolam is around 36.83 mm. The NA = 0.8 is the figure the supplier told me for the condenser, not the objectives. I have attached an image of my Lomo from my initial uni studies in the early 1980s. Notice the brass extensions between the base and the microscope proper. These lift the microscope to allow a ~11 mm closer objective focusing for the absolutely beautiful old Zeiss APO objectives. Notice also the modified stage including very functional mechanical gear not a feature of Biolams. Also the Mitutoyo 1 micron gauge for image stacking convenience.
Attachments
LomoModifiedWeb.JPG
LomoModifiedWeb.JPG (179.86 KiB) Viewed 1796 times

apochronaut
Posts: 2730
Joined: Fri May 15, 2015 12:15 am

Re: Phase contrast microscopy

#4 Post by apochronaut » Wed Apr 03, 2019 12:42 pm

georgetmacro wrote:Hi. I have just purchased an inexpensive Lomo 'negative phase contrast' condenser to experiment with. The NA is 0.8 according to the supplier. The unit comes with two objectives that are supposedly specific to this type of microscopy. The item has not arrived yet so I would like to learn something about the technique beforehand. I sort of know what these instruments do but would like to understand more.
georgetmacro wrote: e.g., why does the higher NA attract a higher price? Is it because the optics are better, bigger?
Objectives with a higher N.A. require a more complex lens system, in order to achieve that N.A. and as well be well corrected for aberrations that those lenses naturally create. You can get a high N.A. with simple lenses but then you need other lenses to compensate for the aberrations or distortions caused by the first lenses. Higher N.A. is desirable because it translates into higher resolving power.
georgetmacro wrote:Why do the instruments require a specific type of objective? I would also like to see any images people have achieved experimenting with the technique. Cheers George.
This answer is pretty much the same as the former but a bit more complex. Whether an objective will work up to it's specifications in any given instrument is dependent on 3 things.
1) will it fit. Generally threads are fairly standard as R.M.S. threads but occasionally a thread is not. Sometimes adapters can be found to fit a desirable objective that has a different mount. Also, even if the objective is the correct thread and fits the conditions of number 2) below, it might be physically too long or short to focus properly in another microscope.
2) Is it for the correct optical system. There are a number of optical systems out there, based on the length from the objective shoulder to the top of the eyepiece tube. Using an objective designed for one optical system in another will result in anything from very little alteration in performance to a completely unusable image. As to knowing whether a given objective will work well enough in another system ? If the system is close enough ; for instance an objective meant for a 160mm tube in a 170mm tube , there will be only a tiny shift in performance. Sometimes there is literature available in order to make that determination or the experience of someone that has tried out various combinations. Often too, you must have a compatible eyepiece.

3)With regards to phase contrast and some other techniques beyond bright field, these situations add a further level of matching components. Phase rings( annuli) becomes a factor in the case of phase contrast. Even if an objective is corrected for the correct optical system, the position, size and composition of the phase annulus in the objective must fit perfectly with the position, size and composition of the phase annulus in the condenser body. The condenser lens system must also be compatible, so it casts the image of the condenser annulus exactly where it is needed in the objective.

Your Lomo system, by the way is probably compatible with some very high quality and not too expensive, Lomo water immersion phase objectives, something few of the Japanese, European or North American systems had available at anything but extremely high prices.

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